Highly packed polycationic ammonium, sulfonium and phosphonium lipids

ABSTRACT

The present invention discloses highly packed polycationic ammonium, sulfonium and phosphonium lipid compounds useful for making lipid aggregates for delivery of macromolecules and other compounds into cells. They are especially useful for the DNA-dependent transformation of cells. Methods for their preparation and use as intracellular delivery agents are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/131,539, filed Apr. 23, 2002, now allowed, which is a division ofU.S. patent application Ser. No. 09/648,492, filed Aug. 25, 2000, nowU.S. Pat. No. 6,399,663, which, in turn, was a division of U.S. patentapplication Ser. No. 09/187,676, filed Nov. 6, 1998, now U.S. Pat. No.6,110,916, which, in turn, was a division of U.S. patent applicationSer. No. 08/782,783, filed Jan. 13, 1997, now U.S. Pat. No. 5,834,439,which, in turn, was a division of U.S. patent application Ser. No.08/171,232, filed Dec. 20, 1993, now U.S. Pat. No. 5,674,908, all ofwhich are incorporated by reference herein to the extent notinconsistent herewith.

FIELD OF THE INVENTION

Highly packed polycationic ammonium, sulfonium and phosphonium lipidcompounds are disclosed, having utility in lipid aggregates for deliveryof macromolecules and other compounds into cells.

BACKGROUND OF THE INVENTION

Lipid aggregates such as liposomes have been found to be useful asagents for delivery to introduce macromolecules, such as DNA, RNA,protein, and small chemical compounds such as pharmaceuticals, to cells.In particular, lipid aggregates comprising cationic lipid componentshave been shown to be especially effective for delivering anionicmolecules to cells. In part, the effectiveness of cationic lipids isthought to result from enhanced affinity for cells, many of which bear anet negative charge. Also in part, the net positive charge on lipidaggregates comprising a cationic lipid enables the aggregate to bindpolyanions, such as nucleic acids. Lipid aggregates containing DNA areknown to be effective agents for efficient transfection of target cells.

The structure of various types of lipid aggregates varies, depending oncomposition and method of forming the aggregate. Such aggregates includeliposomes, unilamellar vesicles, multilamellar vesicles, micelles andthe like, having particle sizes in the nanometer to micrometer range.Methods of making lipid aggregates are by now well-known in the art. Themain drawback to use of conventional phospholipid-containing liposomesfor delivery is that the material to be delivered must be encapsulatedand the liposome composition has a net negative charge which is notattracted to the negatively charged cell surface. By combining cationiclipid compounds with a phospholipid, positively charged vesicles andother types of lipid aggregates can bind DNA, which is negativelycharged, can be taken up by target cells, and can transfect targetcells. (Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA84:7413-7417; Eppstein, D. et al., U.S. Pat. No. 4,897,355.)

A well-known cationic lipid disclosed in the prior art isN-[1-(2,3-dioleoyl-oxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).The structure of DOTMA is:

DOTMA by itself or in 1:1 combination withdioleoylphosphatidylethanolamine (DOPE) is formulated into liposomesusing standard techniques. Felgner, et al. supra demonstrated that suchliposomes provided efficient delivery of nucleic acids to some types ofcells. A DOTMA:DOPE (1:1) formulation is sold under the trade nameLIPOFECTIN (Gibco/BRL: Life Technologies, Inc., Gaithersburg, Md.).Another commercially available cationic lipid is1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), which differsfrom DOTMA only in that the oleoyl moieties are linked via ester, ratherthan ether bonds to the propylamine. DOTAP is believed to be morereadily degraded by target cells. A related group of prior art compoundsdiffer from DOTMA and DOTAP in that one of the methyl groups of thetrimethylammonium group is replaced by a hydroxyethyl group. Compoundsof this type are similar to the Rosenthal Inhibitor (RI) ofphospholipase A (Rosenthal, A. F. and Geyer, R. P. (1960) J. Biol. Chem.235:2202-2206) which has stearoyl esters linked to the propylamine core.The dioleoyl analogs of RI are commonly abbreviated as DORI-ether andDORI-ester, depending on the linkage of the fatty acid moieties to thepropylamine core. The hydroxy group can be used as a site for furtherfunctionalization, for example by esterification to carboxyspermine.

Another class of prior art compounds has been disclosed by Behr et al.(1989) Proc. Natl. Acad. Sci. USA 86:6982-6986; EPO publication 0 394111 (Oct. 24, 1990), in which carboxyspermine has been conjugated to twotypes of lipids. The structure of 5-carboxyspermylglycinedioctadecylamide (DOGS) is:

where R=CH₃(CH₂)₁₇

The structure of dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DPPES) is:

where R=CH₃(CH₂)₁₅

Both DOGS and DPPES have been used to coat plasmids, forming a lipidaggregate complex that provides efficient transfection. The compoundsare claimed to be more efficient and less toxic than DOTMA fortransfection of some cell lines. DOGS is available commercially asTRANSFECTAM™ (Promega, Madison, Wis.).

A cationic cholesterol derivative (DC-Chol) has been synthesized andformulated into liposomes in combination with DOPE. (Gao, X. and Huang,L. (1991) Biochim. Biophys. Res. Comm. 179:280-285) The compound'sstructure is

Liposomes formulated with DC-Chol are said to provide more efficienttransfection and lower toxicity than DOTMA-containing liposomes for somecell lines.

Lipopolylysine, formed by conjugating polylysine to DOPE, has beenreported to be especially effective for transfection in the presence ofserum, a condition likely to be encountered in vivo (Zhou, X. et al.(1991) Biochim. Biophys. Acta 1065:8-14).

Despite advances in the field, a need remains for a variety of improvedcationic lipid compounds. In particular, no single cationic lipid todate has been found to work well with all cell types. Since differentcell types differ from one another in membrane composition, it is notsurprising that different compositions and types of lipid aggregates areeffective for different cell types, either for their ability to contactand fuse with target cell membranes, or for aspects of the transferprocess itself. At present these processes are not well understood,consequently the design of effective liposomal precursors is largelyempirical. Besides content and transfer, other factors are ofimportance, for example, ability to form lipid aggregates suited to theintended purpose, toxicity to the target cell, stability as a carrierfor the compound to be delivered, and ability to function in an in vivoenvironment. In addition, lipid aggregates can be improved by broadeningthe range of substances which can be delivered to cells. The highlypacked and spatially correct positioning of positive charges matchingnegative charges on DNA polycationic ammonium, sulfonium and phosphoniumlipid compounds of the present invention have improved function withrespect to several of the foregoing attributes.

SUMMARY OF THE INVENTION

The present invention provides highly packed polycationic ammonium,sulfonium and phosphonium lipid compounds according to the generalformula:

In the general formula (I),

-   -   X is selected from the group consisting of N, S, P or SO;    -   x is an integer ranging from 1 to about 20;    -   n_(i), where i=1 to x, are, independently of one another,        integers that can have a value ranging from 1 to about 6;    -   R_(A) and R_(B), independently of one another, are selected from        the group consisting of H, or an alkyl, hydroxyalkyl or thiol        substituted alkyl group having from 1 to about 6 carbon atoms;    -   R₁ and R₂, independently of one another, are selected from the        group consisting of alkyl groups having from 1 to about 6 carbon        atoms, where r is either 1 or 0, such that r is 0 or 1 when X is        N, with N being positively charged if r is 1, r is 0 when X is S        or SO, with S and SO being positively charged, and r is 1 when X        is P, with P being positively charged; and    -   A₁-A₂, independently of one another, are selected from the group        consisting of the following groups Z₁-Z₆:    -   Z₁ is a straight-chain alkyl, alkenyl, or alkynyl group having        from 2 to about 22 carbon atoms wherein one or more        non-neighboring —CH₂— groups can be replaced with an O or S        atom;    -   Z₂ is a branched alkyl, alkenyl, or alkynyl group having from 2        to about 22 carbon atoms wherein one or more non-neighboring        —CH₂— groups can be replaced with an O or S atom;    -   Z₃ is a straight-chain or branched alkyl group substituted with        one or two OH, SH, NH₂ or amine groups within about 3 carbon        atoms of the bond between Z₃ and X;    -   Z₄ is a substituted straight-chain or branched alkyl, alkenyl or        alkynyl group having from 2 to about 22 carbon atoms wherein the        substituent is an aromatic, alicyclic, heterocyclic or        polycyclic ring and wherein one or more of the non-neighboring        —CH₂— groups of said alkyl, alkenyl or alkynyl group can be        substituted with an O or S atom.    -   Z₅ is a —B-L group wherein B is selected from the group —CO—,        —CO₂—, —OCO—, —CO—N—, —O—CO—N—, —O—CH₂—, —CH₂—O—, —S—CH₂—,        —CH₂—S— or —CH₂— and L is selected from the group consisting of:    -   Z₁; Z₂; Z₄; or    -   an aromatic, alicyclic, heterocyclic or polycyclic ring moiety;    -   Z₆ is a —CH(D-L)₂ or a —C(D-L)₃ group wherein D is selected from        the group consisting of —CO—, —CO₂—, —OCO—, —CO—N—, —O—CO—N—,        —O—, or —S— and L, is selected from the group consisting of:    -   Z₁; Z₂; Z₄; or    -   an aromatic, alicyclic, heterocyclic or polycyclic ring moiety.

In general in any particular compound of formula I, the chain lengthn_(i) can vary from 1 to 6. For example, in a compound in which x is 3where i is 1 to 3, n₁, n₂ and n₃ can all have the same value, any twocan have the same value or all three can have different values. Inparticular embodiments of the compounds of this invention, the chainlengths n_(i) vary in the repeating pattern 3, 4, 3, 3, 4, 3, . . . .Other particular embodiments of this invention include those in whichn_(i) differ from each other by +/−1.

A groups include those in which two substituents on different X's,preferably neighboring X groups, are covalently linked with each otherto form a cyclic moiety.

The oxygen or sulfur atoms introduced into Z₁ and Z₂ groups arepreferably introduced within about 3 carbon atoms from the bond to the Xgroup.

The aromatic, alicyclic, heterocyclic or polycyclic ring moieties ofthis invention can be substituted or unsubstituted. Substituents includeamong others: OH, SH, NH₂, Ch₃, COCH₃ and halogens, particularly F.Further, one or more of the ring carbons in the alicyclic, heterocyclicor polycyclic ring moieties of this invention can be carbonyl groupsC═O.

Introduction of OH, NH₂ or amine groups as substituents on A groupswithin about 3 carbons from the bond to X can facilitate solubility ofthe compounds of this invention in physiological media.

Compounds of the invention are useful, either alone or in combinationwith other lipid aggregate-forming components (e.g., DOPE, DOSPA, DOTMAor cholesterol) for formulation into liposomes or other lipidaggregates. Such aggregates are polycationic, able to form stablecomplexes with anionic macromolecules, such as nucleic acids. Thepolyanion-lipid complex interacts with cells making the polyanionicmacromolecule available for absorption and uptake by the cell.

Of special interest are the products of general formula (I) in which Xis nitrogen and A₁ and A₂ are Z₁. These N-alkylated-polyamines and theirquaternary ammonium salts are particularly useful for intracellulardelivery of negatively charged macromolecules. This aspect of theinvention is based on the finding that polyamines alkylated with longhydrocarbon chains have enhanced affinity for cells, many of which beara net negative charge, and for various polyanions, such as nucleicacids, relative to the natural polyamine compounds. The effectiveness ofN-alkylated polyamines is thought to result from the increased basicity(easier and stronger protonation) of the secondary and tertiary amines,even under alkaline conditions. Similarly, quarternized polyaminecompounds show increased affinity for negatively charged substances,such as cells and nucleic acids, relative to the natural polyamines. Theincrease in affinity of the quarternized polyamine compounds presumablyresults from the permanent positive charges of the quarternized amines,each aligned in front of a negative charge of the DNA molecule(cooperative effect). Moreover, because of the relatively high lipidcontent of the alkylated and quaternized polyamines, these compoundsalso interact more strongly with the lipid bilayer of cell membranesthan their cognate polyamines.

The combination of high lipid content per molecule and increasedaffinity for anionic macromolecules makes the compounds of the inventionnot only superior intracellular delivery agents, but also less toxic tothe target cells. The reduced toxicity is thought to result in part fromthe fact that a higher binding constant between DNA/lipid leads to alower concentration of lipid needed to completely coat the DNA. Sincetoo much lipid disrupts the cell membrane (these lipids are alsodetergents), a lower amount of lipid should be less toxic. Thisincreased affinity produces a more stable liposome, which in turnincreases the efficiency of delivery. Thus, lower concentrations ofthese highly packed polycationic lipids are required to coat themolecules and bind to the target cells, thereby maximizing efficiency ofdelivery while minimizing cell toxicity.

In addition to the high lipid content and increased affinity for anionicsubstances, the compounds of the present invention also promoteproximity between the complexed polyanion and the target cell membrane,thus increasing interactions between these two entities. In mostliposomal precursors, the lipid moiety is physically separated from thecationic binding site by a relatively large linking group, typicallyranging from five to eight carbon atoms in length. The presentinvention, in contrast, provides straight-chain compounds wherein thelipid constituent is directly attached, without a linker, to thecationic site. The branched compounds of the invention comprise a short(preferably 3 carbon) linker, which enhances the solubility of thesehighly packed lipids. It is believed that the unexpected improvement inefficiency and cell viability observed with these compounds can beattributed, at least in part, to proximity between the liposomalcontents and cell membrane. Although the mechanism is not fullyunderstood, it is believed that less water can intercalate in thisregion, thus minimizing disruption to the cell membrane caused by excesswater.

Of particular interest are the products of general formula (I) in whichX is nitrogen, n is 3 or 4, x is 1, r is 0, R_(A) and R_(B) arehydrogens, and A₁ and A₂ are unbranched alkyl, alkenyl, alkynyl oralkoxy groups having 2 to about 22 carbon atoms.

Most preferred is tetramethyltetrapalmylspermine, the product of generalformula (I) in which X is nitrogen, where x is 3, n₁ is 3, n₂ is 4, andn₃ is 3, R_(A) and R_(B) are hydrogens, R₁ and R₂ are methyl groups, andA₁ and A₂ are unbranched alkyls having 16 carbon atoms.

Specific embodiments of this invention include compounds of formula I inwhich X is N. Of those compounds in which X is N, this inventionincludes, but is not limited to those compounds wherein:

-   -   A₁ and A₂, independently of one another, are selected from the        group Z₁, Z₂, or Z₃;    -   A₁ and A₂, independently of one another, are selected from the        group Z₅; and particularly those Z₅ wherein B is CO, —CH₂—, or        —O—CH₂— and L is Z₁ or Z₂;    -   A₁ and A₂, independently of one another, are selected from the        group Z₆; and particularly wherein D is —O—, —CO—,

—OCO— or —CO₂—;

-   -   R_(A) and R_(B) are H or they are alkyl groups having 1-3 carbon        atoms, inclusive;    -   R₁ and R₂ are alkyl groups having 1 to 3 carbon atoms,        inclusive, and more particularly are methyl groups;    -   n_(i) are all either 3 or 4 and x is 2 to 5;    -   n_(i) alternate in the pattern 3, 4, 3, 3, 4, 3 and x is greater        than or equal to 3; and    -   R_(A) and R_(B) are —CH₂—CH₂—OH groups.

Compounds of this invention of formula I in which X is S include amongothers those in which:

-   -   A₁ and A₂ are R groups which are selected from any of R₅-R₈        where:    -   R5 is a straight-chain (unbranched) alkyl, alkenyl, alkynyl or        alkoxy having 2 to about 22 carbon atoms;    -   R6 is a branched alkyl, alkenyl, alkynyl or alkoxy having 2 to        about 22 carbon atoms;    -   R₇ is an aromatic, alicyclic, heterocyclic or polycyclic ring        moiety; and    -   R₈ is a branched or unbranched substituted alkyl, alkenyl,        alkynyl or alkoxy having from 2 to about 22 carbon atoms,        wherein the substituent is an aromatic, alicyclic, heterocyclic        or polycyclic ring.

Also included in this invention are compounds of formula I where X is Sand x is 1. In this case, R_(A) and R_(B) are preferably methyl groups.Preferred A groups having alkyl, alkenyl alkynyl or alkoxy groups arethose having about 12 to 16 carbon atoms. Preferred R are branched orstraight-chain alkyl, alkenyl or alkynyl groups. In R₅, R₆, and thebranched or straight-chain portion of R₈, one or more non-neighboring—CH₂— groups can be replaced with O or S atoms to give ether orthioether R groups.

Additional subsets of compounds of formula I of this invention includethose in which:

x=1, n is an integer between 2 and 6 inclusive and R is an unbranchedalkyl, alkenyl, alkynyl or alkoxy group having 2 to about 22 carbonatoms; and particularly those in which n is 3;

Wherein at least two of the R groups on different S are covalentlylinked together to produce a cyclic moiety.

In specific embodiments, the compounds of the present invention are alsorepresented by the formulas II and III:

In both formula II and III, R_(A), R_(B), r, Z₁-Z₆ and X are as definedabove for formula I. All of R₁-R₄ can be selected from the groups asdefined above for R₁ and R₂

In formula II:

-   -   n and m, independently of one another, are integers (chain        length) ranging in value from 1 to about 6, with n and m of 3 or        4 being more preferred. It is preferred that the values of m and        n differ only by +/−1;    -   x can be an integer from 1 to 10, with the subset of compounds        having x=2-5 being of particular interest; and    -   A₁-A₃, independently of one another, are selected from the group        Z₁-Z₆, of particular interest are A groups which are        straight-chain alkyl, alkenyl or alkynyl groups.

In formula III:

-   -   l, m, and n, independently of one another, are integers (chain        length) ranging in value from 1 to about 6, with l, m and n of 3        or 4 being more preferred. It is preferred that the values of l,        m and n differ from each other only by +/−1;    -   x can be an integer from 1 to 10, with the subsets of compounds        having x=1, x=2 and x=3-5 being of particular interest; and    -   A₁-A₄, independently of one another, are selected from the group        Z₁-Z₆, with A groups which are straight-chain alkyl, alkenyl or        alkynyl groups of particular interest.

Specific embodiments of this invention include compounds of formulas IIand III in which X is N, m is 3, and n is 4, wherein A₁-A₃,independently of one another, are selected from the groups Z₁, Z₂, andZ₃, and wherein A₁-A₃ are the same group. Other specific embodimentsinclude compounds of formulas II and II in which X is N and A₁-A₃,independently of one another, are selected from the group Z₅, wherein Bis CO, —CH₂—, or —O—CH₂— and L is Z₁ or Z₂. Other embodiments includecompounds of formulas II and III in which X is N and A₁-A₄,independently of one another, are selected from the group Z₆, wherein Dis —O—, —CO—, —OCO— or —CO₂—. Of particular interest are compounds offormulas II and III wherein X is N and R_(A) and R_(B) are H or methylgroups, R₁ and R₂ are methyl groups, m is 3, n is 4, x is 2 to 6. Alsoof particular interest are compounds of formulas II and III wherein X isN and R_(A) and R_(B) are —CH₂—CH₂—OH groups.

Of particular interest are compounds of formula III in which X is N;R_(A) and R_(B) are H; l, m and n are 3 or 4; x is 1; and A₁-A₄,independently of one another, are unbranched alkyl groups having fromabout 12 to 16 carbon atoms. Particularly preferred are compounds offormula III where A₁-A₄ are all the same unbranched alkyl group,particularly an unbranched alkyl group having 16 carbon atoms. Otherembodiments include compounds of formula III wherein X is N; x is 1; l,m and n are 3 or 4; and A₁-A₄ is Z₅, wherein B is CO and L is Z₁,particularly wherein Z₁ has about 12 to about 16 carbon atoms. Otherspecific embodiments include the compounds of formula III wherein X isS; x is 1-5; l, m and n are 2 or 3; and A₁-A₄ is Z₁, particularlywherein A₁-A₄ are unbranched alkyl or alkenyl groups having from 2 toabout 22 carbon atoms, and preferably from about 12 to about 16 carbonatoms. Also of interest are compounds of formula III wherein at leasttwo of the A₁-A₄ are substituents are covalently linked to produce acyclic compound. Other specific embodiments include the compounds offormula III wherein X is P.

In compounds of both formula II and 111, preferred Z groups are branchedor straight-chain alkyl, alkenyl, alkynyl or alkoxy groups, have about12 to about 16 carbon atoms.

This invention also includes compounds having the structure:

wherein Ar₁ and Ar₂ are aryl rings or cyclohexane rings and R isselected from any of R₅-R₈ as defined above. The aryl rings can beselected from among any of phenyl, pyridinyl, pyrimidyl, pyrazinyl,thiadiazole and pyridazinyl. Cyclohexane rings include trans-cyclohexylrings. Of particular interest are compounds of formula IV wherein bothof Ar₁ and Ar₂ are phenyl rings, and wherein R is R₅ or an unbranchedalkyl.

This invention also includes lipid aggregates comprising one or more ofthe compounds of formulas I, II, III or IV or mixtures thereof. Ofparticular interest are lipid aggregates of the compounds of formula I,more particularly those in which X is N.

The transfection methods of the present invention employing compounds offormulas I, II, III or IV or mixtures thereof can be applied to in vitroand in vivo transfection of cells, particularly to transfection ofeukaryotic cells including animal cells. The methods of this inventioncan be used to generate transfected cells which express useful geneproducts. The methods of this invention can also be employed as a stepin the production of transgenic animals. The methods of this inventionare useful as a step in any therapeutic method requiring introducing ofnucleic acids into cells. In particular, these methods are useful incancer treatment, in in vivo and ex vivo gene therapy, and in diagnosticmethods. The transfection compositions of this invention can be employedas research reagents in any transfection of cells done for researchpurposes. Nucleic acids that can be transfected by the methods of thisinvention include DNA and RNA from any source comprising natural basesor non-natural bases, and include those encoding and capable ofexpressing therapeutic or otherwise useful proteins in cells, thosewhich inhibit undesired expression of nucleic acids in cells, thosewhich inhibit undesired enzymatic activity or activate desired enzymes,those which catalyze reactions (Ribozymes), and those which function indiagnostic assays.

The compositions and methods provided herein can also be readily adaptedin view of the disclosure herein to introduce biologically activeanionic macromolecules other than nucleic acids including, among others,polyamines, polyamine acids, polypeptides, proteins, biotin, andpolysaccharides into cells. Other materials useful, for example astherapeutic agents, diagnostic materials and research reagents, can becomplexed by the polycationic lipid aggregates and introduced into cellsby the methods of this invention.

This invention also includes transfection kits which include one or moreof the compounds of formulas I, II, III, IV or mixtures thereof ascationic lipids.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel, highly packed polycationicammonium, sulfonium and phosphonium lipid compounds having uniqueproperties and advantages not heretofore available to the liposome art.The compounds can be used alone or in combination with other compounds(e.g., DOPE, DOTMA and DOSPA) to prepare liposomes and other lipidaggregates suitable for transfection or delivery of compounds other thanDNA to target cells, either in vitro or in vivo.

The novel compounds of general formula (I) are polycationic and thusform highly stable complexes with various anionic macromolecules,particularly polyanions such as nucleic acids. These compounds have theproperty, when dispersed in water, of forming lipid aggregates whichassociate strongly, via their cationic portions, with polyanions. Byusing an excess of cationic charges relative to the anionic compound,the polyanion-lipid complexes may be adsorbed on cell membranes, therebyfacilitating uptake of the desired compound by the cells.

The cationic lipids disclosed herein offer three unique advantages overprior art compounds. First, the compounds of general formula (I)represent novel liposomal precursors wherein the polycationic bindingregions are optimally spaced, preferably equidistance between charges,to provide proper alignment with the anionic phosphates of nucleicacids. Proper alignment of charges increases the binding constant of thelipid to nucleic acid via cooperative interaction. This increasedaffinity produces a more stable DNA-lipid complex, which in turnincreases the efficiency of delivery. Thus, lower concentrations ofthese agents are required to coat the molecules and bind to the targetcells, thereby maximizing efficiency of delivery while minimizing celltoxicity.

The second unique advantage of the compounds disclosed herein is theirunusually high affinity for the lipid bilayer of cell membranes. Unlikethe mono-substituted lipopolyamine compounds currently in use, thecompounds of the invention comprise lipidic substituents at eachcationic binding region. Compounds of the invention thus interact morestrongly with the lipid bilayer of cell membranes than existing cationiclipids. The branched lipids of the present invention comprise multiplelipid moieties per cationic site, and thus bind particularly strongly tocell membranes.

Finally, the compounds of the present invention promote proximitybetween the complexed polyanion and the target cell membrane, thusincreasing interactions between these two entities. Unlike previouscationic lipids, the lipid moieties of the present compounds areattached without linkers to the cationic sites.

Of special interest are the products of general formula (I) in which Xis nitrogen and A₁ and A₂ are Z₁. These N-alkylated-polyamines and theirquaternary ammonium salts are especially useful for intracellulardelivery of negatively charged macromolecules. The unexpectedeffectiveness is thought to result from the increased basicity and thepermanent positive charge of the alkylated and quarternized polyamines,respectively. Moreover, as discussed elsewhere herein, the relativelyhigh lipid content of these compounds is believed to maximizeinteraction with the lipid bilayer of cell membranes. The combination ofhigh lipid content and increased affinity for anionic macromoleculesmakes these compounds not only superior intracellular delivery agents,but also less toxic to the target cells.

In a preferred embodiment of general formula (I), the values of n_(i)are either identical or vary at most by one integer, thus providingapproximately equidistant spacing between the polycationic bindingregions (X). In another preferred embodiment of general formula (I),when A₁ and A₂ are branched hydrocarbons (e.g., Z₂), each branch isconnected to the polymer backbone via a hydrophilic heteroatom such asoxygen, as exemplified herein. The presence of a hydrophilic heteroatommitigates the hydrophobicity of these highly packed compounds, thusenhancing their solubility in water.

Certain of the compounds of this invention may be insufficiently solublein physiological media to employ for delivery and transfection methods.Those of ordinary skill in the art will appreciate that there are avariety of techniques available in the art to enhance solubility of suchcompounds in aqueous media. Such methods are readily applicable withoutundue experimentation to the compounds described herein. As describedherein, one method for increasing solubility of compounds of Formulas Ithrough III is to introduce OH, NH₂, SH, or amine substituents on Zgroups within about 3 carbon atoms from the X group.

The present invention also provides improved methods for transfectingand delivering macromolecules to target cells. The improvement relatesto the use of highly packed polycationic ammonium, sulfonium andphosphonium lipid compounds to either enhance the efficiency of deliveryor to reduce the toxicity to the cells. This invention has significantadvantages over prior art methods which employ neutral or slightly basicdelivery agents having a relatively low lipid content, which interactweakly with both anionic macromolecules and the lipid bilayer of cellmembranes. Because of this limited affinity and low lipid content,current methods require high concentrations of the delivery agent, whichdisrupts cell membranes, often leading to cell death. The presentinvention resolves the problems associated with prior art methods byemploying highly efficient delivery agents which are effective atrelatively low and non-toxic concentrations.

DEFINITIONS

Lipid Aggregate is a generic term which includes liposomes of all typesboth unilamellar and multilamellar as well as micelles and moreamorphous aggregates of cationic lipid or lipid mixed with amphiphaticlipids such as phospholipids.

Target Cell refers to any cell to which a desired compound is delivered,using a lipid aggregate as carrier for the desired compound.

Transfection is used herein to mean the delivery of expressible nucleicacid to a target cell, such that the target cell is rendered capable ofexpressing said nucleic acid. It will be understood that the term“nucleic acid” includes both DNA and RNA without regard to molecularweight, and the term “expression” means any manifestation of thefunctional presence of the nucleic acid within the cell, includingwithout limitation, both transient expression and stable expression.

Delivery is used to denote a process by which a desired compound istransferred to a target cell such that the desired compound isultimately located inside the target cell or in, or on, the target cellmembrane. In many uses of the compounds of the invention, the desiredcompound is not readily taken up by the target cell or appropriatecytoplasmic compartment and delivery via lipid aggregates is a means forgetting the desired compound into the cell cytoplasm.

The polycationic lipids were prepared by following the general reactionschemes given below (Schemes 1-5).

Straight-chain polycationic lipopolyamines were prepared as shown inScheme 1. A polyamine was treated with an acid chloride of the desiredlength in the presence of triethylamine and methylene chloride underargon at room temperature to obtain the corresponding substituted amide(compound 1). Compound 1 was then reduced using lithium aluminum hydridein the presence of anhydrous tetrahydrofurane to give compound 2.Treatment of compound 2 with iodomethane at high temperature yielded apartially quarternized compound (compound 3). Compound 3 was furthermethylated using additional iodomethane to produce the fullyquarternized spermine derivative (compound 4).

Although the above method exemplifies the synthesis ofN,N,N′,N′-tetrapalmylspermine and its quarternized derivatives, thereaction scheme provides a general method for preparing a variety ofN-alkylated and quarternized lipopolyamines. Those of ordinary skill inthe art will appreciate that alternate methods and reagents other thosespecifically detailed herein can be employed or readily adapted toproduce a variety of useful compounds. For example, although the abovereaction scheme uses spermine as the exemplified starting material, thescheme is equally applicable with other straight-chain polyamines.Various polyamines are readily accessible or can be easily synthesizedusing standard methods, for example, by combining acrylonitrile with anappropriate diaminoalkane.

Branched polycationic lipopolyamines are prepared as shown in Scheme 2.A branched amino alcohol is treated with an appropriate nitrile (e.g.,acrylonitrile) to produce a branched amino nitrile of the desired length(compound 4). Compound 4 is then alkylated using 3-bromo-acrylonitrileat high temperature to yield the corresponding dinitrile compound(compound 5). Compound 5 is further alkylated using an alkyl sulfonatein pyrrole to yield compound 6. Reduction of the nitrilo groups ofcompound 6 using lithium aluminum hydride or, alternatively, usinghydrogen in the presence of Raney nickel, yields compound 7. Compound 7is then treated with an acid chloride of the desired length in thepresence of triethylamine and methylene chloride, followed by reductionwith lithium aluminum hydride at high temperature to give compound 8.

Although the above method uses tri-hydroxymethyl-aminomethane as theexemplified starting material, the reaction scheme provides a generalmethod for preparing a variety of branched lipopolyamines. Thesealkylated polyamines can also be quarternized as previously described(see compounds 3 and 4 above) using iodomethane at high temperature.Those of ordinary skill in the art will appreciate that alternatemethods and reagents can be employed or readily adapted to produce avariety of useful branched compounds.

Dicationic sulfonium lipids were prepared as shown in Scheme 3.1,3-propanedithiol was treated with ethylene oxide to afford thecorresponding diol-adduct. The diol was tosilated in the presence ofbase followed by displacement of the tosyl groups by sodium sulfide togive the corresponding mercaptan. This mercaptan was di-alkylated withacetyl bromide using N-butyl lithium as the base. Finally, treating thetetra sulfide with iodomethane affords the sulfonium salt. Polycationicsulfonium lipids were prepared as shown in Scheme 4. Schemes 3 and 4provide a general method for preparing a variety of highly packedsulfonium lipids. Modification and optimization of this method toproduce a variety of useful sulfonium compounds is well within the skillof the ordinary artisan.

Phosphonium lipids were prepared as shown in Scheme 5.Diphenylphosphinic chloride was treated with hydroquinine (excess) toafford the corresponding addition product. Treatment of the additionproduct with sodium hydride followed by acetyl bromide afforded thecorresponding phospholipid. The phospholipid, in turn, is quarternizedwith iodomethane to afford the phosphonium salt.

The compounds of the invention can be used in the same manner as areprior art compounds such as DOTMA, DOTAP, DOGS and the like. Methods forincorporating such cationic lipids into lipid aggregates are well-knownin the art. Representative methods are disclosed by Felgner et al.,supra; Eppstein et al. supra; Behr et al. supra; Bangham, A. et al.(1965) M. Mol. Biol. 23:238-252; Olson, F. et al. (1979) Biochim.Biophys. Acta 557:9-23; Szoka, F. et al. (1978) Proc. Natl. Acad. Sci.USA 75:4194-4198; Mayhew, E. et al. (1984) Biochim. Biophys. Acta775:169-175; Kim, S. et al. (1983) Biochim. Biophys. Acta 728:339-348;and Fukunaga, M. et al. (1984) Endocrinol. 115:757-761. Commonly usedtechniques for preparing lipid aggregates of appropriate size for use asdelivery vehicles include sonication and freeze-thaw plus extrusion.See, e.g., Mayer, L. et al. (1986) Biochim. Biophys. Acta 858:161-168.Microfluidization is used when consistently small (50-200 nm) andrelatively uniform aggregates are desired (Mayhew, E., supra).Aggregates ranging from about 50 nm to about 200 nm diameter arepreferred; however, both larger and smaller sized aggregates arefunctional.

Methods of transfection and delivery of other compounds are well-knownin the art. The compounds of the present invention yield lipidaggregates that can be used in the same processes as those prior artcompounds.

It will be readily apparent to those of ordinary skill in the art that anumber of general parameters are important for optimal efficiency oftransfection or delivery. These parameters include, for example, thepolycationic lipid concentration, the concentration of compound to bedelivered, the medium employed for delivery, the length of time thecells are incubated with the polyanion-lipid complex, and the relativeamounts of cationic and non-cationic lipid. It may be necessary tooptimize these parameters for each particular cell type. Suchoptimization is routine employing the guidance provided herein,including the transfection assays as described in the Examples herein.

It will also be apparent to those of ordinary skill in the art thatalternative methods, reagents, procedures and techniques other thanthose specifically detailed herein can be employed or readily adapted toproduce the liposomal precursors and transfection compositions of thisinvention. Such alternative methods, reagents, procedures and techniquesare within the spirit and scope of this invention.

The preparation and use of representative compounds of the invention arefurther detailed by reference to the following Examples. In each case,the ability of various compounds of the invention to provide efficienttransfection was compared with a control using DOSPA:DOPE (1.5:1 molarratio). All abbreviations used herein are standard abbreviations in theart. Specific procedures not described in detail are either referencedor well-known in the art.

EXAMPLES Example 1 Synthesis of N,N,N′,N′-tetrapalmitoylspermine (1)

To a solution of spermine (0.99 g, 80%, 3.8 mmol) and triethylamine(1.54 g, 15.2 mmol) in methylene chloride (400 ml) at 0° C. under argonwas added palmitoyl chloride (4.17 g, 4.8 ml, 15.2 mmol). The reactioncontinued at room temperature for three days. TLC analysis (silica gel;THF:CH₂Cl₂/1:3) showed a new spot (rf=0.8) and no starting material. Theorganic phase was washed twice with sodium bicarbonate solution (10%,200 ml), hydrochloric acid (1 M, 200 ml) and water (200 ml). Thesolution was dried (Na₂SO₄) and the solvent removed to afford 4 g ofdesired product (90%).

Example 2 Synthesis of N,N,N′N′-tetrapalmylspermine (2)

To a suspension of lithium aluminum hydride (900 mg, 23.7 mmol) inanhydrous tetrahydrofurane (80 ml) was added a solution ofN,N,N′,N′-tetrapalmitoylspermine (500 mg, 0.43 mmol) in anhydroustetrahydrofurane (10 ml). The reaction mixture was refluxed for two daysunder argon. The excess lithium aluminum hydride was removed with sodiumhydroxide (1 M, 5 ml). The organic phase was decanted and the flaskwashed twice with additional tetrahydrofurane (50 ml). The solution wasdried (Na2SO4) and the solvent removed in vacuo to afford almost puredesired tetraamine. This material was passed through a short-path silicagel bed (filtration) and eluted sequentially with ethyl acetate andethyl acetate/triethylamine (10%) to afford, after solvent removal, thedesired product (313 mg, 82%).

Example 3 Synthesis ofN,N,N′,N′-tetramethyltetrapalmyl-sperminetetrammonium iodide (3)

A solution of tetrapalmylspermine (20 mg) in iodomethane (1 ml) washeated at 55° C. for 18 hr. The excess iodomethane was removed in vacuoand the residue redissolved in methylene chloride. This solution wasextracted twice with sodium bicarbonate (3 ml), dried (NaSO₄), and thesolvent removed to afford 25 mg (100%) of desired material.

Example 4 Synthesis ofN,N,N′,N′-hexamethyltetrapalmyl-sperminetetrammonium iodide (4)

A solution of N,N,N′,N′-tetramethyltetrapalmylspermine-tetrammoniumiodide (10 mg) in iodomethane was heated for 48 hr at 80° C. Theiodomethane was removed in vacuo to afford 12 mg of desired product(100%).

Example 5 Lipid Formulation

Tetrapalmylspermine, tetramethylpalmyl-spermine, andhexamethylpalmylspermine were formulated using two protocols. Lipidswere first dissolved in chloroform and aliquots of each lipid with andwithout DOPE were evaporated under vacuum. Lipids were then resuspendeddirectly in water by vortexing, or were suspended first in ethanol (1/10 of final volume) and then diluted with water.

Example 6 Cell Culture and Plasmids

All cell lines were obtained from American Type Culture Collection(Rockville, Md.). Standard tissue culture methods were employed. Chinesehamster ovary (CHO-K1) cells were cultured in EMEM (GIBCO BRL)containing 2 mM proline and 5% (v/v) fetal bovine serum (FBS). NIH 3T3cells were cultured in Dulbecco's-modified Eagle's medium (DMEM)containing 10% (v/v) calf serum (CS). Jurkat cells were cultured in RPMI1640 (GIBCO BRL) containing 10% (v/v) FBS. Human fibroblasts wereisolated from neonatal foreskin tissue and prepared as follows. Rinsed,fresh tissue was exposed to 25 units/ml dispase (Collaborative Research,Bedford, Mass.) overnight at 4° C., separated into epidermis and dermis,and the minced dermis was digested 10 min at 37° C. in 0.25% (v/v)trypsin, 1 mM EDTA. The reaction was stopped by a rinse andcentrifugation in DMEM with 10% (v/v) FBS, in which the cells were alsocultured. All cultures were incubated at 37° C., 5% CO₂. Media for allcultures routinely included 100 units/ml penicillin and 100 μg/mlstreptomycin.

The plasmid vector pCMV β-gal is a commercially available (Clontech,Calif.) mammalian reporter vector containing the E. coli β-galactosidase(β-gal) gene under the control of the Cytomegalovirus promoter (seeMacGregor et al. (1989) Nucleic Acids Res. 17:2365). The plasmid vectorpCMVCAT was also described previously (Boshart et al. (1985) Cell41:521). Plasmid DNA was purified by standard cesium chloride methods.

Example 7 Transfection of CHO-K1, NIH-3T3, and Human Fibroblast Cells

For transfection of CHO-K1, NIH-3T3, and human fibroblast cells in24-well plates, lipid and DNA (pCMV β-gal) were diluted separately into25 μl aliquots of Opti-MEM I Reduced Serum Medium (GIBCO BRL;serum-free). These aliquots were gently mixed and incubated at roomtemperature for 15-45 minutes to form lipid-DNA complexes. The complexeswere added to cells in each well containing 250 μl serum-free growthmedium. Cells were exposed to DNA-lipid complexes for 4-5 hr understandard culture conditions, after which 1 ml normal growth medium wasadded. Antibiotics were never present during lipid-mediatedtransfections. At 24 hr after transfection, cells were assayed in situfor β-galactosidase activity.

Example 8 Transfection of Jurkat cells

For transfection of Jurkat cells in suspension, lipid and DNA (pCMVCAT)were diluted separately into 500 μl aliquots of Opti-MEM I Reduced SerumMedium (GIBCO BRL; serum-free). These aliquots were gently mixed andincubated at room temperature for 15-45 minutes to form lipid-DNAcomplexes. For each transfection sample, 1×10⁶ Jurkat cells werecentrifuged in a microfuge tube. The cell pellets were suspended withthe lipid-DNA complex solutions and transferred to wells of 12-wellplates. Cells were exposed to DNA-lipid complexes for 4-5 hr understandard culture conditions, after which 0.5 ml growth medium containing30% FBS, 150 μg/ml Phorbol myrystate acetate (PMA; Sigma Chemical Co.,St. Louis, Mo.), and 3 μg/ml phytohemagglutinin (PHA) were added to afinal concentration of 10% FBS, 50 μg/ml PMA and 1 μg/ml PHA,respectively. After approximately 24 hr, one ml growth medium containing10% FBS, 50 ng/ml PMA and 1 μg/ml PHA was added to each well.Antibiotics were never present during lipid-mediated transfections.Cells were harvested at approximately 48 hr post-transfection bycentrifugation. Cell lysates were prepared by resuspending cell pelletsat 0° C. in 1 M Tris-HCl pH 8.0 containing 0.1% Triton X-100 and 5 μlaliquots were assayed for CAT activity.

Example 9 Transient Transfection Assays

Cell lysates were assayed for β-galactosidase activity as described bySanes et al. (1986) EMBO J. 5:3133. Cells were rinsed with PBS, fixedfor 5 minutes in 2% (v/v) formaldehyde, 0.2% glutaraldehyde in PBS,rinsed twice with PBS, and stained 2 hr to overnight with 0.1% β-gal, 5mM potassium ferrocyanide, 5 mM potassium ferrocyanide, 2 mM MgCl₂ inPBS. Rinsed cells were photographed using a 10× or 20× objective on aNikon inverted microscope with Hoffman optics. Transfection efficiencyis evaluated by counting or estimating the number of β-gal positivecells (blue-stained cells).

Cell lysates were assayed for CAT activity as described by Neumann etal. (1987) BioTechniques 5:444. Cells were rinsed with PBS and frozen at−70° C. in 0.5-1.5 ml 0.1% Triton X-100 in 0.1 M Tris, pH 8.0. Afterrapid thawing at 37° C., the lysate was cleared by centrifugation. Whenmore than 5 μl of a 1 ml extract was to be used in the assay, theextract was heated to 65° C. for 10 minutes to inactivate anydeacetylases present, and centrifuged again. When necessary, the extractwas diluted in 0.1 M Tris pH 7.8-8.0. Lysate was incubated with 50 nCi¹⁴C Butyryl CoA (New England Nuclear, Boston, Mass.) and 0.25 μMoleschloramplienicol in 0.1 M Tris, pH 7.8-8.0, in a total volume of 0.25 mlin a 4-ml scintillation vial. Reaction mixtures were incubated at 37° C.for 2 hr, overlaid with 3 ml Econofluor (Dupont, Boston, Mass.),inverted once and then incubated for an additional 2 hr at roomtemperature before counting. Each assay included a standard curve of0.001 to 0.05 units CAT enzyme (Pharmacia, Uppsala, Sweden), the linearrange for these reaction conditions. Extracts were diluted or volumeswere adjusted in order to have activity within the linear range duringthe assay. To insure accuracy, the activity was normalized to the samevolume per transfection.

Example 10 Results

Results are shown in Tables 1-4. The “CAT ACTIVITY” column in Table 4indicates the relative transfection effectiveness of the lipidformulation in Jurkat cells. The control sample was a cell culture grownunder similar conditions as those that were transfected, but with no DNAor cationic lipid added.

For primary human fibroblast cells (Table 1), NIH-3T3 cells (Table 2),CHO-K1 cells (Table 3), and Jurkat cells (Table 4), compound 3 washighly effective for DNA transfection with minimal toxicity. Data inTables 1-4 show that compound 3 has efficiency comparable to DOSPA forthe transfection of human fibroblasts and Jurkat cells, but a lowerconcentration of lipid is required for optimal activity. When using lowamounts of DNA in NIH-3T3 cells, compound 3 was approximately 10-foldmore effective than DOSPA.

TABLE 1 TRANSFECTION RESULTS WITH PRIMARY HUMAN FIBROBLASTS LipidOptimal Lipid β-gal Positive (Molar ratio) Conc. (μg) Cells (app. %)Compound 3:DOPE (1:1) 1 5 DOSPA:DOPE (1.5:1) 5 5 Cells were plated in24-well plates at a density of 3 × 10⁴ cells per well. The followingday, cells in each well were transfected with a suboptimal concentration(200 ng) of pCMV β-gal DNA, using the indicated lipid formulations.

TABLE 2 TRANSFECTION RESULTS WITH NIH-3T3 Lipid Optimal Lipid β-galPositive (Molar ratio) Conc. (μg) Cells (app. %) Compound 3:DOPE (1:1) 110 DOSPA:DOPE (1.5:1) 4 1 Cells were plated in 24-well plates at adensity of 4 × 10⁴ cells per well. The following day, cells in each wellwere transfected with a suboptimal concentration (200 ng) of pCMV β-galDNA, using the indicated lipid formulations.

TABLE 3 TRANSFECTION RESULTS WITH CHO-K1 Lipid Optimal Lipid β-galPositive (Molar ratio) Conc. (μg) Cells (app. %) Compound 3:DOPE (1:1)1-1.2 20 DOTMA:DOPE (1:1) 1.5 10-15 DOSPA:DOPE (1.5:1) 3-5   80-90 Cellswere plated in 24-well plates at a density of 6 × 10⁴ cells per well.The following day, cells in each well were transfected with 200 ng ofpCMV β-gal DNA, using the indicated lipid formulations.

TABLE 4 TRANSFECTION RESULTS WITH JURKAT CELLS Lipid Optimal Lipid CATActivity (Molar ratio) Conc. (μg) (mUnits CAT/5 μl) Compound 3:DOPE 619.8 DOTMA:DOPE (1:1) 5 5.0 DOSPA:DOPE (1.5:1) 25 19.2 Cells (1 × 10⁶)were transfected with 2 μg of pCMVCAT DNA as described above, using theindicated lipid formulations. At 48 hr post-transfection, cell lysateswere prepared and 5 μl aliquots were assayed for CAT enzyme activity.

1. A compound or polycation having the formula:

or salt thereof where: x is an integer ranging from 1 to about 20; 1 isan integer ranging from 1 to about 6; r and s, independently of oneanother, are 0 or 1, wherein when r is 1, the N bonded to R₁ and A₁ hasa positive charge, and when s is 1, the N bonded to R₂ and A₂ has apositive charge; R_(A) and R_(B), independently of one another, areselected from the group consisting of H, or an alkyl, hydroalkyl orthiol-substituted alkyl group having from 1 to 6 carbon atoms; R1 andR2, independently of one another, are selected from the group consistingof alkyl groups having 1 to about 6 carbon atoms; and A1 and A2,independently of other A1 and A2 groups, are selected from the groupconsisting of a —CH(D-L)₂ and a —C(D-L)₃ group wherein D is selectedfrom the group consisting of —CO—, —CO₂—, —O—C—O—, —CO—N—, —O—CO—N—,—O—, and —S—, and L is selected from the group consisting of: (a) astraight chain or branched alkyl, alkenyl, or alkynyl group having from2 to about 22 carbon atoms wherein one or more non-neighboring—CH₂—groups can be replaced with an O or S atom; (b) a substituted straightchain or branched alkyl, alkenyl, or alkynyl group having from 2 toabout 22 carbon atoms wherein the substituent is an aromatic, alicyclicheterocyclic or polycyclic ring and wherein one or more of thenon-neighboring neighboring —CH₂— groups of said alkyl, alkenyl oralkynyl group can be substituted with an O or S atom; and (c) anaromatic, alicyclic, heterocyclic and a polycyclic ring moiety.
 2. Alipid aggregate which comprises one or more compounds of claim
 1. 3. Amethod for delivery of a macromolecule to a cell comprising the step ofcontacting said cell with a composition which comprises the compound ofclaim 1 and said macromolecule.
 4. A compound or polycation having theformula:

or salt thereof where: x is an integer ranging from 1 to about 20; 1 isan integer ranging from 1 to about 6; r and s, independently of oneanother, are 0 or 1, wherein when r is 1, the N bonded to R₁ and A₁ hasa positive charge, and when s is 1, the N bonded to R₂ and A₂ has apositive charge; R_(A) and R_(B), independently of one another, areselected from the group consisting of H, or an alkyl, hydroalkyl orthiol-substituted alkyl group having from 1 to 6 carbon atoms; R1 andR2, independently of one another, are selected from the group consistingof alkyl groups having 1 to about 6 carbon atoms; and A1 and A2,independently of other A1 and A2 groups, are selected from the groupconsisting of a B-L group wherein B is selected from the groupconsisting of —CO—, —CO₂—, —O—C—O—, —CO—N—, —O—CO—N—, —O—CH₂—, —S—CH₂—,—CH₂—S—, and —CH₂— and L is selected from the group consisting of: (a) astraight chain or branched alkyl, alkenyl, or alkynyl group having from2 to about 22 carbon atoms wherein one or more non-neighboring—CH₂—groups can be replaced with an O or S atom; (b) a substituted straightchain or branched alkyl, alkenyl, or alkynyl group having from 2 toabout 22 carbon atoms wherein the substituent is an aromatic, alicyclicheterocyclic or polycyclic ring and wherein one or more of thenon-neighboring neighboring —CH₂— groups of said alkyl, alkenyl oralkynyl group can be substituted with an O or S atom; and (c) anaromatic, alicyclic, heterocyclic and a polycyclic ring moiety.
 5. Amethod for delivery of a macromolecule to a cell comprising the step ofcontacting said cell with a composition which comprises the compound ofclaim 4 and said macromolecule.
 6. A compound or polycation having theformula:

or salt thereof where: x is an integer ranging from 1 to about 20; 1 isan integer ranging from 1 to about 6; r and s, independently of oneanother, are 0 or 1, wherein when r is 1, the N bonded to R₁ and A₁ hasa positive charge, and when s is 1, the N bonded to R₂ and A₂ has apositive charge; R_(A) and R_(B), independently of one another, areselected from the group consisting of H, or an alkyl, hydroalkyl orthiol-substituted alkyl group having from 1 to 6 carbon atoms; R1 andR2, independently of one another, are selected from the group consistingof alkyl groups having 1 to about 6 carbon atoms; and A1 and A2,independently of other A1 and A2 groups, are selected from the groupconsisting of a substituted straight chain or branched alkyl, alkenyl,or alkynyl group having from 2 to about 22 carbon atoms wherein thesubstituent is an aromatic, alicyclic heterocyclic or polycyclic ringand wherein one or more of the non-neighboring neighboring —CH₂— groupsof said alkyl, alkenyl or alkynyl group can be substituted with an O orS atom.
 7. A method for delivery of a macromolecule to a cell comprisingthe step of contacting said cell with a composition which comprises thecompound of claim 6 and said macromolecule.
 8. A compound or polycationhaving the formula:

or salt thereof where: x is an integer ranging from 1 to about 20; 1 isan integer ranging from 1 to about 6; r and s, independently of oneanother, are 0 or 1, wherein when r is 1, the N bonded to R₁ and A₁ hasa positive charge, and when s is 1, the N bonded to R₂ and A₂ has apositive charge; R_(A) and R_(B), independently of one another, areselected from the group consisting of H, or an alkyl, hydroalkyl orthiol-substituted alkyl group having from 1 to 6 carbon atoms; R1 andR2, independently of one another, are selected from the group consistingof alkyl groups having 1 to about 6 carbon atoms; and A1 and A2,independently of other A1 and A2 groups, are selected from the groupconsisting of a straight chain or branched alkyl, alkenyl, or alkynylgroup having from 2 to about 22 carbon atoms wherein one or more of thenon-neighboring neighboring —CH₂— groups of said alkyl, alkenyl oralkynyl group can be substituted with an O or S atom.
 9. A method fordelivery of a macromolecule to a cell comprising the step of contactingsaid cell with a composition which comprises the compound of claim 8 andsaid macromolecule.
 10. A compound or polycation having the formula:

or salt thereof where: x is an integer ranging from 1 to about 20; 1 isan integer ranging from 1 to about 6; r and s, independently of oneanother, are 0 or 1, wherein when r is 1, the N bonded to R₁ and A₁ hasa positive charge, and when s is 1, the N bonded to R₂ and A₂ has apositive charge; R_(A) and R_(B), independently of one another, areselected from the group consisting of H, or an alkyl, hydroalkyl orthiol-substituted alkyl group having from 1 to 6 carbon atoms; R1 andR2, independently of one another, are selected from the group consistingof alkyl groups having 1 to about 6 carbon atoms; and A1 and A2,independently of other A1 and A2 groups, are selected from the groupconsisting of a straight chain or branched alkyl group substituted withone or two SH groups within about 3 carbon atoms of the bond between A1or A2 and N.
 11. A method for delivery of a macromolecule to a cellcomprising the step of contacting said cell with a composition whichcomprises the compound of claim 10 and said macromolecule.